The term “menotropins” is applied to a hormonal combination of gonadotropins obtained from menopausal and post-menopausal women's urine comprising two glycoprotein hormones: follicle stimulating hormone (FSH) and lutenizing hormone (LH); they are secreted by the pituitary gland, subsequently metabolized, and excreted in the urine.
Urinary gonadotropins have been used successfully to stimulate the ovaries in ovulation induction cycles or during assisted reproduction for several decades.
Since their introduction up to now, there have been significant improvements in manufacturing technology and purification processes. Due to the appropriate application of advanced technology in purification, it has become possible to obtain gonadotropins with a high purity grade, as for example HMG-HP.
European Patent EP1169349 B1 relates to gonadotropin compositions, particularly to FSH (follicle stimulating hormone: follitropin) and menotropin compositions of high biological activity and to a method for preparing these compositions from human crude urine of menopausal and postmenopausal women. The process disclosed by said European Patent EP1169349 B1 allows obtaining compositions of HMG-HP comprising potencies of FSH of 3700 IU/mg protein and 4300 IU/mg protein in examples 1 and 2 respectively of EP1169349 B1.
Despite these advances, urinary products still contains some non-gonadotropin proteins as contaminants (Bassett et al, Analytical identification of additional impurities in urinary-derived gonadotrophins 2009).
Some of the main contaminants of the gonadotropin HMG-HP composition comprise plasma serine protease inhibitor, afamin, insulin-like growth factor binding protein 7, zinc alpha-2-glycoprotein and albumin; all of them identified in the publication Bassett et al, 2009).
The challenge for improving the purification of urinary HMG-HP was to remove those proteins which co-purified with active principles but preserving the integrity of the FSH and LH bioactivities using a simple process with high yield.
To achieve this goal a new purification step was introduced to optimize the purification of HMG-HP of the prior art.
Briefly, during the HMG-HP manufacturing process as presented in the previous patent (European patent EP1169349 B1), two important fractions are obtained in the last chromatographic step (Hydrophobic interaction chromatography, HIC): one of them with FSH bioactivity (Fraction J2) and the other with LH bioactivity (Fraction J3).
Menotropin is a preparation that requires to have a FSH:LH bioactivity ratio of about 1:1.
To achieve a FSH:LH ratio of 1:1, there is a balance step to adjust the content of both hormones. In the case of HMG-HP, this balancing step is done by mixing FSH bioactivity from Fraction J2 with the LH bioactivity from Fraction J3.
As described in the current patent, Fraction J3 is usually contaminated with non-active proteins.
Therefore, to obtain the HMG-UP (HMG ultrapure) material without almost any detectable protein contamination, the LH bioactive fraction (Fraction J3) was successfully purified by multimodal chromatography, specifically a multimodal strong anion exchanger, Capto-adhere (Fraction J3-UP).
As it is well-known (ASRM Practice Committee; Fertility and Sterility, 90, Suppl. 3, S13, 2008) a small amount of hCG present in the preparation contributes to give the majority of the LH bioactivity of menotropin. Consequently, the purification of Fraction J3 implied the purification of the human chorionic gonadotropin (hCG) present in this fraction to obtain Fraction J3-UP.
Finally, to produce HMG-UP, the balancing step involved in this case mixing in equivalent amount the FSH bioactivity from Fraction J2 with LH bioactivity from the now much more purified Fraction J3-UP instead of the previous more contaminated Fraction J3.
For obtaining a composition of HMG-UP, the solution obtained after the balancing step is subjected to the steps of sterile filtration, nanofiltration and precipitation.